Send Orders of Reprints at [email protected] Current Bioactive Compounds 2012, 8, 000-000 1 Thymoquinone: Major Molecular Targets, Prominent Pharmacological Actions and Drug Delivery Concerns Anjali Singha, Iqbal Ahmada, Sohail Akhtera, Mohammad Zaki Ahmadc, Zeenat I. Khana,b and Farhan J. Ahmada,b* a Nanomedicine Lab, Jamia Hamdard, New Delhi 110062, India; bDepartment of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India; cDreamz College of Pharmacy, Khilra-Meramesit, Sundernagar, Himachal Pradesh-36, India Abstract: The oil of black seed (Nigella sativa) has been used as a folk medicine to treat a number of physiological disorders such as epilepsy, gastric problems, allergic conditions and various hepato-biliary ailments. Despite being unaware of the underlying phenomena for the cure of these diseases, people especially in the Mediterranean region have been using this ancient herb since ages. Researches in the 20th century conducted with the aim of tracking molecular pathways of anti-oxidant and anti-inflammatory mechanisms disclosed a great deal of information pertaining to the structural-activity relationships. Rigorous studies revealed that the major active constituent of Nigella sativa oil (NSO) is Thymoquinone (THQ) which exerts the majority of pharmacological actions observed by the essential oil. Since then, a large number of investigator groups have identified numerous molecular pathways of almost all major diseases which can be cured by THQ. Being a phytochemical, THQ has a high lipophilicity which makes it a poorly soluble agent in aqueous fluids restricting its systemic bioavailability. Also, THQ is highly light and heat-sensitive which further complicates its successful formulation for drug delivery. This review thus offers an insight to adequately comprehend the large number of pharmacological actions along with general mechanisms and major molecular targets pertaining to THQ. Additionally, a section dealing with the application of nanotechnological approaches to appropriately deliver the drug to its intended site of action has been elaborated. Keywords: Anti-cancer, molecular targets, nanoparticles, nanotechnology, thymoquinone. INTRODUCTION Nature has surrounded the mankind and animal kingdom with a diversity of plants and herbs. Scientific findings have discovered the power of healing present in plants which can be attributed to the active chemical moiety existing in various parts. Some of these chemical groups possessing significant pharmacological properties include polyphenols (Resveratrol), flavonoids, steroids (Digoxin) and alkaloids (Vincristine). A rather big class of chemical agents are the quinones which occur widely in gymnosperms and angiosperms of which some commonly encountered ones include benzoquinones (Thymoquinone), naphthoquinones (Lawsone) and anthraquinones (Sennosides). One such therapeutically important benzoquinone is Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) (THQ). Botanically, THQ is found in the seeds of the annual herb Nigella sativa belonging to the family Ranunculaceae containing upto 30-48% of THQ along with p-cymene (7– 15%), carvacrol (6–12%), 4-terpineol (2–7%), t-anethole (1– 4%) and sesquiterpene longifolene (1–8%) [1]. Other active principles isolated from N. sativa are hydrothymoquinone, polythymoquinone, nigellicine, nigellidine, nigellimine-Noxide, thymol and alpha-hedrin. This herb has been held in *Address correspondence to this author at the Associate Professor, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India; Tel: +919810720387; Fax: 01126059663; E-mail: [email protected] 1573-4072/12 $58.00+.00 great respect by the Arab population and popularly called as the “seed of blessing”. Illnesses such as asthma, bronchitis, rheumatism and various infections can be cured by the seed oil. In addition, NSO has also been used to treat skin conditions, such as eczema and boils. Major pharmacological properties shown by the oil include anti-epileptic, antihypertensive, anti-histaminic, anti-diabetic, anti-inflammatory, anti-microbial, anti-oxidant and anti-cancer [2]. The US-FDA has kept the black seed on its GRAS list. The antioxidant activity of THQ is mainly credited to its potent radical scavenging action, especially against superoxide anions. Indirectly, THQ exerts its anti-oxidant action by significantly enhancing the transcription of genes responsible for the production of body’s natural anti-oxidant enzymes like SOD (super-oxide dismutase), CAT (catalase), and Glutathione peroxidase (GSH-Px) [3]. Most of the studies elaborating on the actions of NSO have been performed on in-vitro cell cultures or by administering either aqueous suspensions, decoctions or oily suspensions to animal models. Majority of drug in aqueous/oily suspension formed via oral route is either prematurely metabolized or improperly absorbed. Thus, there is a need to suitably develop an effective and highly bioavailable formulation of THQ to completely realize its potential as a drug for treatment of various maladies. The formulability of THQ is often a difficult task and poses a great deal of challenge to the formulation scientist. It has an extremely lipophilic character (log P = 2.54) showing © 2012 Bentham Science Publishers 2 Current Bioactive Compounds 2012, Vol. 8, No. 3 poor formulation characteristics into a conventional dosage forms such as tablets/capsules. Further, the dissolution process is also hampered due to its high hydrophobicity. Another important problem is that of its thermo-labile nature which renders it unsuitable to a number formulation approaches including nano-formulation techniques. Hence, a proper and strategic fabrication using novel methods of nanotechnology can prove as a potential solution towards bioavailability enhancement and target oriented delivery of THQ to various organs of the body. Thus, in this review, we attempt to highlight the major molecular targets of THQ along with the nanotechnological advancements that have been accomplished to overcome bioavailability and targeting related glitches of this ancient herb. Furthermore, the possibilities of exploring other arenas of nanotechnology for incorporation and delivery of THQ have also been elaborated as future prospects. MAJOR MOLECULAR TARGETS Anti-oxidant Activity Extensive literature is present glorifying the potent antioxidant activity of THQ in-vitro and in-vivo [1,4]. Major reason for the development of oxidative stress that is, the imbalance between the levels of oxidants and physiological anti-oxidant machinery, is due to the chain of events triggered by the ROS (Reactive oxygen species) such as superoxide ions or nitrite ions. Extensive oxidative stress ultimately culminates to chronic inflammatory disorders which further worsen and lead to the development of tumors as well as cardiovascular, neurological and hepato-renal ailments. Major molecular targets which are modulated by THQ are numerous and conformingly established. A brief discussion of these molecular targets is described below which not only helps to understand the basic mechanism of action THQ but also aids in designing nano-therapeutics with an enhanced targeting effect at the site of action. A schematic elaboration of the major mechanism of THQ action is presented in (Fig. 1). (a). Free radical scavenging action: A considerable free radical and superoxide radical scavenging action at nano- and micro-molar concentrations is exerted by THQ. Furthermore, it has been found to inhibit irondependent lipid peroxidation which varies as the dose is increased [5]. Additionally, a superior superoxide anion scavenging activity of THQ was observed when compared to tert-butylhydroquinone (TBHQ). Inhibition of p44/42 and p-38 proteins constitutes another mechanism by which THQ suppresses the transcription of nitric oxide synthase (NOS) and the ultimate production of nitric oxide (NO), which in turn forms peroxynitrite upon combination with a superoxide. This peroxynitrite causes tissue injury by nitration of tyrosine residues [6]. An important illustration of this effect is represented by the abolishment of doxorubicin-associated cardiotoxicity by THQ without compromising with its anti-tumor activity, caused mainly due to the production of ROS [7]. (b). Induction of synthesis of anti-oxidant enzymes: Antioxidant enzymes counter the free radicals produced by internal and external factors. THQ is found to enhance the mRNA expression of the genes controlling the pro- Singh et al. duction of anti-oxidant enzymes such as superoxidedismutase (SOD), catalase (CAT), glutathione peroxidase (G-Px) and glutathione-S-peroxidase. This fact has been supported by the study conducted in diethylnitrosomine induced rat model [8]. Also, quinone reductase (QR) and glutathione transferase (GT) production is enhanced by THQ as observed by Nagi and Almakki in mice liver [9]. (c). Enhanced mitochondrial function: Substrate utilization and/or oxidative phosphorylation are significantly promoted by THQ along with a concomitant increase in the production of ATPs which ultimately lead to greater energy production and mitochondrial function [10]. Anti-inflammatory Actions Protective effects of THQ elicited via reduction in Interleukins (IL-1) and Tumor necrosis factor (TNF-
) in adjuvant-induced arthritis support the obvious role in interrupting inflammatory responses [11,12]. Moreover, ovalbumin induced asthmatic mouse model displayed elevated levels of Leukotrienes (LT-B4), C4, Th2 cytokines and eosinophils in bronchoalveolar lavage fluid. THQ challenged all these actions and additionally suppressed 5-LOX (Lipoxygenase) intimately associated with airway inflammation. Multifunctional nuclear transcription factor (NF-B) has also been found to be inhibited by THQ. El-Gazzar et al reported the induction of repressive NF- B p50 homodimer binding to the promoter in LPS-induced (Lipopolysaccharide) RBL2H3 rat basophil cells [13,14]. However, a different point of view was expressed by Sethi et al who reported that NF- B inhibition is caused due to the inhibition of TNF-induced IB
degradation and phosphorylation along with p65 translocation [15]. Besides, constitutive and IL-6 induced STAT3 phosphorylation in U266 multiple myeloma cells have been found to be inhibited by THQ along with c-Src and JAK-2 activation. The study further disclosed the interplay of cyclin D1, Bcl-2, Bcl-xL, surviving, Mcl-1 and VEGF in the phosphorylation of U266 cells. Anti-cancer Targets (a). Cell cycle arrest: Induction of G0/G1 arrest in mouse papilloma carcinoma cells via augmented expression of p16 and decrease in cyclin D1, in acute lymphoblastic leukemia Jurkat cell line through p73-dependent pathway as well as in HCT116 human colorectal carcinoma cells through p53 regulation has been observed by THQ administration [16]. In addition, G1 to S phase progression in LNCaP prostate cancer cells, G2/M arrest in mouse spindle carcinoma cells, in MNNG/HOS human osteosarcoma cells and in MCF-7/DOX doxorubicin resistant breast cancer cells was also demonstrated by THQ [17]. (b). Pro-apoptotic effects: THQ induces apoptosis in p-53 dependent and independent pathway, HCT116 human colorectal carcinoma cells, p-53 mutant MNNG/HOS osteosarcoma cells, p-53 null myeloblastic leukemia HL-60 cells by caspase 8, 9, 3 activation and PC-3 prostate cancer cells to name a few targets. pTEN and STAT3 are also involved in the apoptotic effects of THQ with a significant decrease in p-Akt [18]. Other Thymoquinone Current Bioactive Compounds 2012, Vol. 8 No. 3 3 Fig. (1). Schematic representation of major mechanism of action of thymoquinone targets include TNF-induced NF-
B regulated gene products including IAP1, IAP2, XIAP, Bcl-2, survivin, COX-2, MMP-9 and VEGF [19]. Apoptosis induction in FG/COLO357 pancreatic cancer cells while downregulation of mucin-4 through proteasomal pathway forms another mechanism of the pro-apoptotic effects of THQ [20,21]. brain ailments to cardiovascular diseases to cancer abrogation mediated via a complex interaction with nuclear proteins (Fig. 2). (c). Anti-proliferative effects: THQ has exhibited the inhibition of proliferation in mouse neoplastic keratinocytes including glioma/glioblastoma (U87 MG and T98G, M059K and M059J), breast adenocarcinoma (multidrug-resistant MCF-7/TOPO, MCF-7, MDA-MB-231 and BT-474), leukemia (HL-60 and Jurkat), lung cancer (NCI-H460 and A549), colorectal carcinoma (HT-29, HCT-116, DLD-1, Lovo and Caco-2), pancreatic cancer (MIA PaCa-2, HPAC and BxPC-3), osteosarcoma (MG63 and MNNG/HOS), prostate cancer (LNCaP, C42B, DU145 and PC-3) [22-27]. Apart from these, a large number of targets are also regulated by THQ but they do not have convincing evidence. Only the major targets that have been sufficiently elaborated by scientists in animal and human models in-vitro and invivo have been presented above. PHARMACOLOGICAL ACTIONS OF THQ Being an extremely useful therapeutic moiety, THQ has proven its worth in a large number of animal studies from Fig. (2). Major areas of proven thymoquinone activity 4 Current Bioactive Compounds 2012, Vol. 8, No. 3 Neurological Disorders Structural, biochemical or electrical abnormalities in the brain, spinal cord or other nerves can result in a range of symptoms such as paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness. THQ has been employed in natural as well as formulated forms to treat a variety of brain maladies. Hosseinzadeh and Parvardeh showed the efficacy of intra-cerebroventricularly administered THQ to counter pentylene tetrazole (PTZ) and maximal electroshock (MES) induced seizure models along with effects on pentobarbital induced hypnosis, locomotor activity. Results showed intraperitoneally administered THQ (40 and 80mg/kg) reduced the duration of mycolinic seizures and prolonged their onsets. Furthermore, THQ impaired the motor co-ordination and decreases locomotor activity implying that an increase in opioid receptor mediated GABA action is responsible for its anti-convulsant activity in petitmal epilepsy [28]. In yet another study, Ilhan et al established the antioxidant and antiepileptic activity of nigella sativa oil (NSO) on PTZ induced kindling seizure in mice. They concluded that NSO exhibited its superior action in attenuating and suppressing PTZ induced oxidative injury as compared to valproate. This action could be elicited by the potent inhibition of ROS formation by THQ via a variety of mechanisms as described in the previous section [29]. Ethanol induced apoptotic neurodegeneration in prenatal rat cortical neurons was attempted to treat with metformin and THQ separately and synergistically. 100mM ethanol exposure for 12h triggered neuronal death via activation of multiple stress pathways. Metformin and THQ enhanced the cell viability as well reduced the elevation of cytosolic free calcium and normalized mitochondrial trans-membrane potential. They concluded that this effect could be attributed to the decreased expression of Bcl-2 (anti-apoptotic protein), increased Bax and cytochrome-C suggesting the alteration of the expression of some key proteins such as Bcl-2, Bax, Caspase-9, Caspase-3 and PARP-1 by the administration of THQ and Metformin [30]. Minimization of sodium valproate induced hepatotoxic implications was reported by Raza et al. They described that THQ in PTZ as well as MES models increased the efficacy of sodium valproate at doses 50 and 100mg/kg. When THQ was coadministered with valproate in drinking water for 21 days, significant reduction in serum ALT (Alanine transferase) and AST (Aspartate transaminase), non-protein sulfhydryls and increased lipid peroxidation in hepatocytes was observed [31]. A pilot study consisting of 22 children with refractory epilepsy were treated with 1mg/kg THQ as adjunctive therapy for 4 weeks. The reduction in frequency of seizures was deduced in the group treated with THQ along with a standard anti-epileptic drug statistically as well as by parental satisfaction [32]. Gastroenterological and Renal Disorders The efficacy of NSO in treating acute gastric ulcer rat models was established by Arsalan et al. They presented a decrease in lipid peroxidation due to malonaldehyde and SOD and the recovery of glutathione content was achieved by THQ (20mg/Kg) in ethanol induced gastric damage and proposed that the effect could be due to the antioxidant property of THQ [33]. Nagi and Almakki proposed that the pro- Singh et al. tective action of THQ in mice liver could be mediated due to the induction of detoxifying enzymes including quinone reductase (QR) and glutathione transferase (GT). In their study, THQ (1, 2 and 4mg/kg/day p.o) administration for 5 days produced a marked increase in the activity of QR (147, 196 and 197% of control, respectively) and GT (125, 152 and 154% of control, respectively). Oral administration of THQ thus makes it a potential prophylactic agent against chemical toxicity [9]. D-galactosamine induced liver injury and corresponding altered hepatic function was used as a model to demonstrate the hepatoprotective effect of THQ in rats. THQ pre-treatment owing to its potent antioxidant action produced a marked reduction in elevated serum enzymes and hepatic malonaldehyde levels almost comparable to that of Silymarin. Histopathological examinations further proved the recovery of damaged liver lobular architecture by THQ. The authors concluded the evidence of membrane stabilizing effect of THQ in protecting liver enzyme leakage and lipid peroxidation [34]. Mansour et al. demonstrated the effects of orally administered THQ on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase activity in hepatic, cardiac and kidney tissues of mice. They determined that THQ (25, 50 and 100mg/kg/day) given for 5 successive days produced significant decrease in hepatic SOD, CAT, GSHPx while cardiac SOD activity and lipid peroxidation were decreased at higher doses (50 and 100mg/kg/day). On the other hand, an increase in DT-diaphorase activity was observed in cardiac and renal tissues with increase in THQ dose. These reports were backed by the combinatorial antioxidant properties of THQ and its metabolite DHTHQ (Dihydrothymoquinone) [35]. Hepatotoxic model of CCl4 treated mice was administered a single dose THQ (100mg/ kg). CCl4 induced liver damage along with elevated serum ALT was significantly (p<0.001) reversed by THQ [36]. THQ and DHTHQ, dose dependently, inhibited nonenzymatic lipid peroxidation induced Fe3+-ascorbate in liver homogenate and DHTQ showed a superior action as compared to THQ and BHT [36]. In a similar study, acetaminophen induced hepatoxic mice were treated with three doses of THQ (0.5, 1 and 2mg/kg/day) for 5 days orally. A considerable and dose dependent reduction in serum ALT activities, total nitrite/nitrate, lipid peroxides, reduced glutathione and increase in ATP was observed. The authors conclusively reported the significant resistance of THQ to oxidative and nitrosative stress along with an improvement in the mitochondrial energy production [6]. Renal ischemia is a complex physiological condition characterized by failure of both the kidneys and renal allografts. The efficacy of THQ against renal ischemia/reperfusion in rat models was fruitfully demonstrated by [37]. NSO administered showed pronounced decrease in serum blood urea nitrogen, malonaldehyde, nitric oxide, protein carbonyl content and creatinine while an increase in SOD, CAT, GSH-Px and total antioxidant capacity was also observed. A beneficial effect on hepatic enzymes in streptozocinnicotinamide induced diabetic rat models has been shown by THQ in a rather recent study conducted by Pari and Sankaranarayanan [38]. They showed that intragastrically administered THQ (20, 40, 80 mg/kg) for 45 days dosedependently improved the levels of insulin and haemoglobin Thymoquinone Table 1. Current Bioactive Compounds 2012, Vol. 8 No. 3 5 Nanotechnological Advancements for the Delivery of Poorly Soluble Herbal Bio-actives Bio-active Herbal Compound Nano-formulation Method of Preparation Outcome References PLGA-nanoparticles Nano-precipitation Enhanced suppression of proliferation of colon cancer, breast cancer, prostate cancer and multiple myeloma [68] Molecular micelles of modified PLGA nanoparticles emulsion solvent evaporation More effective than free THQ against MDA-MB-231 cancer cell growth inhibition [69] amphiphilic biodegradable coreshell nanoparticles emulsification solvent evaporation Controlled release behaviour and augmented cytotoxicity on prenatal rat neuronal hippocampal and fibroblast cells [70] Thermosensitive (NIPAAm-VP) co-polymeric micelles radical co-polymerization Hundred times more efficient against S.aureus, B.subtilis and E.coli strains [71] Capsaicin Nanoemulsion Self-assembly emulsification Improved stability and pharmacokinetics [76] Silymarin Pro-liposomes film-deposition on carriers Increased oral bioavailability, stability and gastro-intestinal absorption [77] Camptothecin Solid lipid nanoparticles High pressure homogenization Sustained release, dose reduction and decreased systemic toxicity [78] Vinpocetine Solid lipid nanoparticles ultrasonic-solvent emulsification Improved absorption and oral bioavailability [79] Paclitaxel (PTX) Carbon nanotubes conjugating PTX to branched PEG chains on single-walled carbon nanotubes via a cleavable ester bond Higher efficacy in suppressing tumor growth than clinical Taxol in a murine 4T1 breast cancer model [72] Piperine Nanospheres homogenization followed by ultrasonication Higher bioavailability and low volume of distribution [80] Resveratrol Nanosuspension High pressure homogenization Enhanced dermal penetratiom [81] Safranal Nano-liposomes Fusion and homogenization Enhanced dermal penetration [82] Podophyllotoxin Multi-walled CNTs Encapsulation Improved targeting and drug stability [83] Thymoquinone with accompanying decrease in glucose and HbA1c. The study thus establishes the potential application of THQ (80 mg/kg) as an anti-hyperglycaemic agent following its normalizing effects on carbohydrate metabolizing enzymes. Cardiovascular Diseases Attia et al put forth the hypolipidemic properties of THQ on reactive oxygen species, anti-oxidant activity and steatosis in livers of hyperlipidemic rabbits. THQ supplementation (20 mg/kg/day) to high cholesterol fed rabbits showed a positive effect upon the serum glucose, insulin, and aminotransferases when compared to the control groups, confirming the attenuation of oxidative stress in fatty liver disease caused by high cholesterol diets in rabbits [39]. A study of the protective effects of the propolis and THQ on atherosclerosis formation in cholesterol fed rabbits was undertaken by Nader et al. Enhanced serum total cholesterol, trigylcerides lipoprotein-cholesterol thiobarbituric acid-reactive substances and decreased HDL-cholesterol was countered by both propolis and THQ along with beneficial effects on kidney function parameters [40]. Studies conducted on hypocholesterolemic effect of thymoquinone rich fraction (TQRF) extracted from Nigella sativa seeds using supercritical fluid extraction (SFE) in comparison with commercial available THQ in male Sprague–Dawley rats were investigated for over 8 weeks. Results showed Plasma total cholesterol levels (TC) and low density lipoprotein cholesterol (LDLC) were significantly decreased in the TQRF and TQ treated rats compared to untreated rats. mRNA level of low density lipoprotein receptor (LDLR) was significantly expressed and the mRNA level of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-COAR) was significantly suppressed in the TQRF and TQ treated rats at different doses compared to untreated rats. This path breaking study identifies TQRF and TQ as cholesterol lowering agents via the uptake of LDLC through up regulation of LDLR gene and inhibition of the synthesis of cholesterol via suppressing the HMG-COAR gene [41]. 6 Current Bioactive Compounds 2012, Vol. 8, No. 3 Antimicrobial Effects Kouidhi et al. demonstrated the MICs (Minimum inhibitory concentrations) of THQ, tetracycline and benzalkonium chloride and conducted the efflux assay of 4,6-diamidino-2phenylindole (DAPI) to determine the effect of THQ on DAPI cell accumulation. Considerable antimicrobial activity was exerted by THQ resulting in a fourfold potentiation of the tested antibiotics and antiseptics. THQ additionally inhibited DAPI efflux activity resulting in its increased accumulation in clinical isolates and decreased loss from bacteria thus, establishing the resistance-modifying action of THQ on oral bacteria [42]. Noteworthy anti-microbial activity of Tunisian Nigella sativa against S. mitis, S. mutans, S. constellatus and G. haemolysins was observed upon the administration of essential oil containing 3.35
g of THQ. The IC-50 of THQ was found to be 19.25+-1.6
g/ml. The study also disclosed an important finding that THQ exerted a more pronounced inhibitory effect on human epithelial cell lines (Hep-2) as compared to the essential oil delineating its anticariogenic activity against bacterial infections [43]. Kamel et al proved the utility of THQ in preventing bacterial biofilm formation. A biofilm is described as the community of cells attached to a biotic or abiotic surface. Minimum biofilm inhibition concentration achieved using crystal violet assay revealed that anti-oxidant activity of cells was modulated by THQ supplementation. The study was carried out on 11 human pathogenic bacteria such as Gram positive cocci Staphylococcus aureus ATCC 25923 (22
g/ml) and Staphylococcus epidermidis CIP 106510 (60
g/ml) [44]. Eman Halawani performed a series of experiments to determine the anti-microbial activity of THQ against Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri, Salmonella Typhimurium, Salmonella Enteritidis and Staphylococcus aureus. S. aureus was found to be highly susceptible to THQ while it was observed that THQ required to inhibit and kill S. aureus was 400 and 800 μg/ml, respectively i.e 100 times more than that of THQ [45]. Combinatorial therapy of THQ and dihydrothymoquinone with anti-biotics such as ampicillin, cephalexin, chloramphenicol, tetracycline and gentamicin could exert synergism in therapy against S. aureus. Only a handful of studies are available in literature which encompass the anti-fungal activity of THQ. One such research was carried out by Aljabre et al on eight species of dermatophytes. Anti-fungal capacity of NSO, THQ and griseofulvin was compared and concluded that NSO and THQ had much greater MIC than griseofulvin in-vitro [46]. The antirhinosinusitis potential of THQ was explored by Cingi et al. They induced rhinosinusitis by intra-nasally administering platelet-activating factor. The histopathological results proved the promising potential of THQ for the treatment of rhinosinusitis and proving the effect to be equivalent to an antibiotic [47]. Anti-cancer Properties Anti-inflammatory actions of THQ can be most conveniently cited as the major underlying anticancer mechanisms since 20% of all human cancer in adults are attributed to chronic inflammatory state or have an inflammatory etiology [48]. Numerous in-vitro studies have shown the remarkable anti-tumor effect of THQ on cancer cell lines such as A549 (lung carcinoma), HEp-2 (larynx epidermoid carcinoma), Singh et al. HT-29 (colon adenocarcinoma) and MIA PaCa-2 (pancreas carcinoma) [49] as well as canine osteosarcoma (COS31), its cisplatin-resistant variant (COS31/rCDDP), human breast adenocarcinoma (MCF7), human ovarian adenocarcinoma (BG-1) and Madin-Darby canine (MDCKcell lines) [50]. Furthermore, a rather recent study has described the antitumor effects of THQ, Doxorubicin and equimolar mixture of both against HL-60 leukemia, 518A2 melanoma, HT-29 colon, KB-V1 cervix, and MCF-7 breast carcinomas as well as their multi-drug-resistant variants and non-malignant human fibroblasts (HF). The anti-apoptotic effects were mainly exerted by DNA fragmentation, caspase-3, -8 and -9, changes in the mitochondrial membrane potential and the ratio of the mRNA expressions of pro- and anti-apoptotic proteins bax and bcl-2 [51]. Not much study has been carried out on the beneficial effects of THQ in brain tumors. Gurung et al tested the anti-cancer effects of THQ on human glioblastoma and normal cells. They summarised that the glioblastoma cells were found to be more sensitive to THQ which induced DNA damage, cell cycle arrest and apoptosis. Also, THQ facilitated telomere breakage by inhibiting the activity of telomerase which signals towards the role of DNA-PKcs. Telomeres in glioblastoma cells with DNAPKcs were more sensitive to THQ mediated effects as compared to those cells deficient in DNA-PKcs [22]. Moreover, THQ has been found to reduce the nephrotoxicity associated with cisplatin [52]. The significant reductions in serum urea and creatinine as well as improvement in polyuria, kidney weight, and creatinine clearance confirm the studies along with the histopathological evidence. Fibrosarcoma and Leukemia Badary and Gamal El-Din et al. in their experiment discovered the lethal effect of THQ in fibrosarcoma animal model. Additionally, a parallel decrease in hepatic lipid peroxides and enhanced GSH, GST and QR levels was also reported delineating chemopreventive as well as therapeutic potential of THQ [53]. Identification of the anti-apoptotic and epigenetic integrator UHRF1 (Ubiquitin-like, containing PHD and RING Finger domains, 1) by the effect of THQ on p-53 deficient acute lymphoblastic leukemia Jurkat cell line was undertaken, since knockdown of p73 expression restores UHRF1 expression. This study thus establishes the subsequent targeting of UHRF1 in p-53 mutated cells via p-73 dependent pathway in acute lymphoblastic leukemia [54,55]. Pancreatic and Gastric Cancer Chehl et al has confirmed the protective and superior effect of THQ in pancreatic ductal adenocarcinoma cells via inhibition of pro-inflammatory cytokines and chemokines such as monocyte chemo-attractant protein-1, TNF-, IL-1 and COX-2 as compared to specific HDAC inhibitor trichostatin A [56]. Evidence of inhibitory action of THQ on redox sensitive cancer target NF-B has been established by [57]. The reported potential role of THQ in down-regulating gemcitabine and oxyplatin induced NF-B activation in pancreatic cancer cells proving the synergistic worth of THQ in gemcitabine and oxyplatin antitumor therapy. The direct down-regulation of MUC4 by THQ has given a new dimension to the treatment of pancreatic cancer using phytochemicals. Incubation of MUC4 expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF with THQ indicated the Thymoquinone downregulation of MUC4 via proteasomal pathway and apoptosis induction in cancerous cells by activation of p-38 mitogen-activated protein kinase pathways [58]. For the first time, the chemosensitizing potential of THQ and 5-fluorouracil (5-FU) was discovered on gastric cancer cells both in-vitro and in-vivo. THQ was found to potentiate the anti-apoptotic activity of 5-FU via downregulation of bcl-2, up-regulation of bax and activation of caspase-6 and caspase-9 and thus chemosensitizes gastric cancer cells to 5FU induced cell death [59]. Inhibitory effect of THQ against benzo-pyrene induced fore-stomach carcinogenesis was investigated by [60]. THQ reduced dramatically benzopyreneinduced fore-stomach tumor occurrence and multiplicity by 70% and 67%, respectively. Substantial accumulation of lipid peroxide, decreased glutathione (GSH) content, glutathione-S-transferase (GST) and DT diaphorase activities were observed in the liver of tumor-bearing mice owing to the strong anti-oxidant behaviour of THQ. Breast and Lung Cancer Enhanced PPAR-  activity along with down-regulation of the expression of bcl-2, bcl-xL and survivin genes in breast cancer was established by suggesting the probable action of THQ as a ligand of PPAR-. Thus, the involvement of this novel pathway by THQ can be utilized as an efficient system to specifically target breast cancer [23]. The ability of THQ to inhibit doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation has also been reported. It arrested the MCF-7/DOX cells at G2/M phase which is attributable to the PTEN expression alteration and elevation of PTEN mRNA. Moreover, THQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins [17]. Radiosensitizing activity of THQ on human breast carcinoma cells (MCF7 and T47D) has been reported by [61]. A supra-additive cytotoxic effect was exerted by THQ when administered in combination with a single dose of ionizing radiation (2.5 Gz) measured by cell proliferation and colony-formation assays. The combined potential of THQ and Cisplatin was realized by Jafri et al. THQ induced apoptosis in both NCI-H460 and NCI-H146 cell lines and inhibited the invasion and reduced the production of two cytokines ENA-78 and Groalpha involved in neo-angiogenesis. Additionally, THQ downregulated NF-B expression which to a certain extent may contribute to overcome cisplatin resistance in lung cancer cells [62]. The ability of THQ to cause necrosis and apoptosis in HEp-2 human laryngeal carcinoma cells has well been established by Rooney and Ryan [63]. They further identified the role of THQ in enhancing cancer cell apoptosis upon GSH depletion induced by buthionine sulfoximine (BSO), a selective inhibitor of glutathione (GSH) synthesis. Prostate and Colorectal Cancer Kaseb et al performed studies concerning the prevention of hormone-refractory prostate cancer by using THQ. Invitro and in-vivo studies implied the crucial role of THQ in inhibiting proliferation and sustainability of cancerous (LNCaP, C4-B, DU145, and PC-3) but not noncancerous (BPH-1) prostate epithelial cells. This seems to be mediated by the down-regulation of Androgen Receptor (AR) and Current Bioactive Compounds 2012, Vol. 8 No. 3 7 E2F-1 (regulated cell proliferation and viability). The study conducted on mice divulged no traces of side effects on proliferation and viability of androgen-sensitive as well as androgen-independent prostate cancer cells [64]. Yi et al. divulged the in-vitro, in-vivo blockage of angiogenesis in xenograft human prostate cancer (PC3) model in mouse by THQ with almost no chemotoxicity. Further, the greater susceptibility of endothelial cells signaled the inhibition of VEGF-induced extra-cellular kinase activation and thus, outlining the therapeutic effect against tumor angiogenesis and growth [65]. Gali-Muhtasib et al compared the apoptosis of THQ in p53+/+ and p53
/
colon cancer cells HCT116. A noticeable up-regulation in p53
/
cells of survival gene CHEK1 was witnessed in response to THQ due to the lack of transcriptional repression of p53. This study hence aids in understanding the contribution of the inhibition of the stress response sensor CHEK1 to the cytotoxic activity of specific DNA-damaging drugs such as THQ. In a yet another study, Gali-Muhtasib et al discussed the utility of THQ in C26 mouse colorectal carcinoma spheroids. THQ injected intraperitoneally (i.p.) suggestively reduced the numbers and sizes of ACF (aberrant crypt foci) at week 10 signalling the potential use of THQ as a therapeutic agent in human colorectal cancer [66]. DRUG DELIVERY CONCERNS AND NANOTECHNOLOGICAL PERSPECTIVES The universal drawbacks of using phytochemical agents for therapeutic purposes are their poor aqueous solubility and subsequent absorption in the body, non-targeted drug distribution as well as low safety margin. Application of nanotechnological approaches can significantly control the drug release and the therapeutics index by enhancing localization in target tissues. Nanoformulation based tactics (1100 nm in at least one dimension) can significantly increase the dissolution of poorly soluble drugs, in addition to augmenting their stability, bioavailability and decreasing their toxicity. Furthermore, surface modification and functionalization of nanocarriers can be utilized for effective targeting of the drug to the specific sites of action. Some nanotechnological developments for the delivery of poorly soluble herbal bio-actives and their outcomes are presented in table 1. Rationally designed nanocarriers with controlled key properties such as size, surface characteristics and the targeting moiety can revolutionize the use of phytochemicals for prophylaxis, diagnosis and treatment of various ailments. Reduction of particles to nano range alters the biodistribution characteristics of the formulation as well. Nanoparticles often cross anatomical barriers by passing through small fenestrations present in the cell lining of each organ which is distinct in its own way. For example, blood brain barrier is intact and has no fenestrations while rat liver has passages of upto 230 nm in their endothelial lining [67]. Thus, a crucial and intellectual control of nanoparticle size can enhance the targetability of the drugs to certain extent. THQ represents one such class of modern phytochemicals which has been extensively studied and pathways of its molecular mechanisms have been comprehensively elucidated. Utilization of these molecular targets coupled with suitable nanotech- 8 Current Bioactive Compounds 2012, Vol. 8, No. 3 nological approaches can work wonders for the treatment of neurological, gastroenterological disorders and various oncological conditions as described above. A rather different study concerning with THQ-conjugates undertaken by Breyer et al outlined the synthesis of 4-Acylhydrazones and 6-alkyl derivatives of THQ for growth inhibition of human HL-60 leukemia, 518A2 melanoma, KB-V1/Vbl cervix, and MCF-7/Topo breast carcinoma cells. The presence of a double bond provided better activities as compared to equal length saturated chains. The 6-hencosahexaenyl conjugate emerged to be the most effective in all resistant tumor cells with an IC50 of 30 nM in MCF-7/Topo cells [68]. This proves the involvement of different mechanisms for tumor suppressive action which causes a superior inhibitory effect. A large number of phytochemicals are extremely hydrophobic in nature and when tested out of the animal model domain, they simply fail to elicit the therapeutic properties in human clinical trials due to safety issues and bioavailability matters. Nanoparticles basically encompass nano-crystals, nanopowders and nano-clusters which are currently areas of intense research of researchers globally. In the recent past, a study dealing with the fabrication of polymeric nanoparticles of THQ has been published. In this experiment conducted by Ravindran et al poly(lactide-co-glycolide) (PLGA) nanoparticles using PEG 5000 as stabilizer by nanoprecipitation technique were formulated. The synthesized nanoparticles showed a particle size varying from 150-200nm and entrapment efficiency of 97.5%. These THQ-NPs were found to be more potent than THQ in suppressing the proliferation of colon cancer, breast cancer, prostate cancer and multiple myeloma [69]. In another study, molecular micelles of THQ using modified PLGA nanoparticles were prepared using the emulsion solvent evaporation method employing anionic molecular micelles as emulsifiers. These THQ loaded nanoparticles proved to be more effective than free THQ against MDA-MB-231 cancer cell growth inhibition with cell viability of 16±5.6% after 96h [70]. Shah et al, synthesized amphiphilic biodegradable core-shell nanoparticles by emulsification solvent evaporation method. Diblock copolymers were prepared by chemical coupling of poly(3hydroxybutyrate-co-3-hydroxyvalerate) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) to monomethoxy-PEG through trans esterification. In vitro cytotoxicity was undertaken on prenatal rat neuronal hippocampal and fibroblast cells showing the capability of amphiphilic nanoparticles to act as safe carriers for the controlled release of THQ [71]. Thermosensitive N-Isopropyl acryl amide-N-Vinyl 2pyrollidone (NIPAAm-VP) co-polymeric micelles were created by radical co-polymerization entrapping the extract of Nigella sativa to check the release and evaluate its antibacterial activity. The effectiveness of the loaded extract was evaluated using gram positive strain of Staphylococcus aureus, Bacillus subtilis and a gram negative strain of Escherichia coli. The polymeric micelles were found to be hundred times more efficient in comparison to the naked ones [72]. SLNs (Solid lipid nanoparticles) originated as an alternative to the polymeric nanoparticles in the 1990’s owing to the increasing mammalian cell toxicity reported by scientists worldwide. SLNs essentially consist of a solid lipid core stabilized by a surfactant and/or co-surfactant to form an aqueous dispersion. NLCs or nanostructured lipid carriers are novel and superior formulations aimed at maximizing Singh et al. drug loading into the SLNs. NLCs usually employ a combination of lipids either solid or liquid to entrap drug molecules. A greater asymmetry in lipid crystal lattices implies more space for incorporation of drug particles as compared to single lipid formulations. The relevance of this concept can be realized when a large dose of drug is to be employed. Both SLNs and NLCs systems can be efficiently applied for the better entrapment, improved drug loading as well as controlled release of hydrophobic drugs like THQ in desired dose. Talking of nanoemulsions (NE), they can be used as modified drug delivery tools for poorly water-soluble drugs. They provide as a reservoir of drug from which the release can be controlled for achieving prolonged and enhanced therapeutic effect and can be described as a thermodynamically stable isotropically clear dispersion of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules. The dispersed phase usually consists of small particles or droplets, with a size range of 5100 nm having a very low oil/water interfacial tension. NEs show versatility with respect to route of administration and can be delivered via parentral, oral, topical, ocular or pulmonary routes. Zulli et al attempted to encapsulate ubiquinone into nanoemulsions with a view to enhance its oral and topical bioavailability. Ubiquinone-NE and ubiquinone were compared for their activity in cell cultures against the synthesis of collagen I and the activity of mitochondria and their resistance against stress of dermal fibroblasts and keratinocytes [73]. Undoubtedly, ubiquinone-NE revealed a significant increase in the bioavailability and thus, therapeutic efficacy. On similar lines, THQ can also be encapsulated into NEs for enhancement of bioavailability. Restricted solubility in gastric fluid can also be overcome by intelligent and strategic development of self-nano/micro emulsifying drug delivery systems (SNEDDS/SMEDDS) of hydrophobic drugs [74]. Two important studies concerning SNEDDS have been undertaken using curcumin as model drug. In the first study, using ethyl oleate as the oil and poloxamer 188, Cremophor®, tween 80 and propylene glycol as surfactants, Cui et al, 2009 enhanced the solubility of curcumin to 21 mg/ml in the SMEDDS. 16 fold higher bioavailability of curcumin was achieved by formulation of SMEDDS [75]. These researches point towards the successful utilization of SNEDDS/SMEDDS for bioavailability enhancement of lipophilic agents including phytochemicals. Carbon nanotubes (CNTs) are a bumper discovery for drug delivery especially in the targeted treatment of cancer. They have currently garnered ample interest due to their ability to alter drug delivery through bio-sensing methods. In one such study conducted by Liu et al, synthesised paclitaxel (PTX) loaded CNTs by conjugating PTX to branched PEG chains on single-walled carbon nanotubes (SWNTs) via a cleavable ester bond to obtain a water-soluble SWNT-PTX conjugate. In-vivo administration of this conjugate led to higher efficacy in suppressing tumor growth than clinical Taxol in a murine 4T1 breast cancer model [76]. This effect was most likely observed due to the prolonged blood circulation and 10-fold higher tumor PTX uptake by SWNT delivery through enhanced permeability and retention effect (EPR). Owing to the immense potential of THQ in the treatment and prophylaxis of various neoplasms, its suitable de- Thymoquinone Current Bioactive Compounds 2012, Vol. 8 No. 3 9 Fig. (3). Thymoquinone delivery concerns and their possible nanotechnological solutions livery using CNTs can lead to a paradigm shift in its utilization. Some of the key nanocarriers with brief specification and their applicability in terms of THQ are illustrated in (Fig. 3). ACKNOWLEDGEMENTS Declared none. REFERENCES CONCLUSION Ideally, minimization of toxicity to non-target sites and maximization of drug concentration at the site of action for the achievement of full therapeutic efficacy is the goal of nanotechnology. Researches have evidently clarified that perfect utilization of a drug can be achieved by its proper spatial and temporal placement in the body. THQ as an effective treatment of various neurological, cardiovascular, gastroenterological disorders and carcinomas can be transformed into a nanocarrier based system for the enhancement of bioavailability as well as efficacy. Moreover, nanotechnological approaches will certainly explore new arenas and potentials of THQ as a versatile bioactive. Presently, animal toxicological data presents THQ as a safe molecule but it is imperative to establish the safety of THQ when incorporated into particular nanocarriers intended for delivery to animal or human subjects. 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Revised: August 31, 2012 Accepted: October 22, 2012